First Three-Dimensional Structure of <i>Toxoplasma gondii</i> Thymidylate Synthase–Dihydrofolate Reductase: Insights for Catalysis, Interdomain Interactions, and Substrate Channeling

Most species, such as humans, have monofunctional forms of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) that are key folate metabolism enzymes making critical folate components required for DNA synthesis. In contrast, several parasitic protozoa, including Toxoplasma gondii, contain a unique bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) having the catalytic activities contained on a single polypeptide chain. The prevalence of T. gondii infections across the world, especially for those immunocompromised, underscores the need to understand TS-DHFR enzyme function and to find new avenues to exploit for the design of novel antiparasitic drugs. As a first step, we have solved the first three-dimensional structures of T. gondii TS-DHFR at 3.7 Å and of a loop truncated TS-DHFR, removing several flexible surface loops in the DHFR domain, improving resolution to 2.2 Å. Distinct structural features of the TS-DHFR homodimer include a junctional region containing a kinked crossover helix between the DHFR domains of the two adjacent monomers, a long linker connecting the TS and DHFR domains, and a DHFR domain that is positively charged. The roles of these unique structural features were probed by site-directed mutagenesis coupled with presteady state and steady state kinetics. Mutational analysis of the crossover helix region combined with kinetic characterization established the importance of this region not only in DHFR catalysis but also in modulating the distal TS activity, suggesting a role for TS-DHFR interdomain interactions. Additional kinetic studies revealed that substrate channeling occurs in which dihydrofolate is directly transferred from the TS to DHFR active site without entering bulk solution. The crystal structure suggests that the positively charged DHFR domain governs this electrostatically mediated movement of dihydrofolate, preventing release from the enzyme. Taken together, these structural and kinetic studies reveal unique, functional regions on the T. gondii TS-DHFR enzyme that may be targeted for inhibition, thus paving the way for designing species specific inhibitors

Design, Synthesis, and Antiviral Evaluation of Chimeric Inhibitors of HIV Reverse Transcriptase

In a continuing study of potent bifunctional anti-HIV agents, we rationally designed a novel chimeric inhibitor utilizing thymidine (THY) and a TMC derivative (a diarylpyrimidine NNRTI) linked via a polymethylene linker (ALK). The nucleoside, 5′-hydrogen-phosphonate (H-phosphonate), and 5′-triphosphate forms of this chimeric inhibitor (THY-ALK-TMC) were synthesized and the antiviral activity profiles were evaluated at the enzyme and cellular level. The nucleoside triphosphate (<b>11</b>) and the H-phosphonate (<b>10</b>) derivatives inhibited RT polymerization with an IC₅₀ value of 6.0 and 4.3 nM, respectively. Additionally, chimeric nucleoside (<b>9</b>) and H-phosphonate (<b>10</b>) derivatives reduced HIV replication in a cell-based assay with low nanomolar antiviral potencies

Temporal Resolution of Autophosphorylation for Normal and Oncogenic Forms of EGFR and Differential Effects of Gefitinib

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases (RTK). EGFR overexpression or mutation in many different forms of cancers has highlighted its role as an important therapeutic target. Gefitinib, the first small molecule inhibitor of EGFR kinase function to be approved for the treatment of nonsmall cell lung cancer (NSCLC) by the FDA, demonstrates clinical activity primarily in patients with tumors that harbor somatic kinase domain mutations in EGFR. Here, we compare wild-type EGFR autophosphorylation kinetics to the L834R (also called L858R) EGFR form, one of the most common mutations in lung cancer patients. Using rapid chemical quench, time-resolved electrospray mass spectrometry (ESI-MS), and Western blot analyses, we examined the order of autophosphorylation in wild-type (WT) and L834R EGFR and the effect of gefitinib (Iressa) on the phosphorylation of individual tyrosines. These studies establish that there is a temporal order of autophosphorylation of key tyrosines involved in downstream signaling for WT EGFR and a loss of order for the oncogenic L834R mutant. These studies also reveal unique signature patterns of drug sensitivity for inhibition of tyrosine autophosphorylation by gefitinib: distinct for WT and oncogenic L834R mutant forms of EGFR. Fluorescence studies show that for WT EGFR the binding affinity for gefitinib is weaker for the phosphorylated protein while for the oncogenic mutant, L834R EGFR, the binding affinity of gefitinib is substantially enhanced and likely contributes to the efficacy observed clinically. This mechanistic information is important in understanding the molecular details underpinning clinical observations as well as to aid in the design of more potent and selective EGFR inhibitors

Illuminating the Molecular Mechanisms of Tyrosine Kinase Inhibitor Resistance for the FGFR1 Gatekeeper Mutation: The Achilles’ Heel of Targeted Therapy

Human fibroblast growth factor receptors (FGFRs) 1–4 are a family of receptor tyrosine kinases that can serve as drivers of tumorigenesis. In particular, <i>FGFR1</i> gene amplification has been implicated in squamous cell lung and breast cancers. Tyrosine kinase inhibitors (TKIs) targeting FGFR1, including AZD4547 and E3810 (Lucitanib), are currently in early phase clinical trials. Unfortunately, drug resistance limits the long-term success of TKIs, with mutations at the “gatekeeper” residue leading to tumor progression. Here we show the first structural and kinetic characterization of the FGFR1 gatekeeper mutation, V561M FGFR1. The V561M mutation confers a 38-fold increase in autophosphorylation achieved at least in part by a network of interacting residues forming a hydrophobic spine to stabilize the active conformation. Moreover, kinetic assays established that the V561M mutation confers significant resistance to E3810, while retaining affinity for AZD4547. Structural analyses of these TKIs with wild type (WT) and gatekeeper mutant forms of FGFR1 offer clues to developing inhibitors that maintain potency against gatekeeper mutations. We show that AZD4547 affinity is preserved by V561M FGFR1 due to a flexible linker that allows multiple inhibitor binding modes. This is the first example of a TKI binding in distinct conformations to WT and gatekeeper mutant forms of FGFR, highlighting adaptable regions in both the inhibitor and binding pocket crucial for drug design. Exploiting inhibitor flexibility to overcome drug resistance has been a successful strategy for combatting diseases such as AIDS and may be an important approach for designing inhibitors effective against kinase gatekeeper mutations

Crystal Structures of HIV‑1 Reverse Transcriptase with Picomolar Inhibitors Reveal Key Interactions for Drug Design

X-ray crystal structures at 2.9 Å resolution are reported for two complexes of catechol diethers with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles

Picomolar Inhibitors of HIV Reverse Transcriptase Featuring Bicyclic Replacement of a Cyanovinylphenyl Group

Members of the catechol diether class are highly potent non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). The most active compounds yield EC₅₀ values below 0.5 nM in assays using human T-cells infected by wild-type HIV-1. However, these compounds such as rilpivirine, the most recently FDA-approved NNRTI, bear a cyanovinylphenyl (CVP) group. This is an uncommon substructure in drugs that gives reactivity concerns. In the present work, computer simulations were used to design bicyclic replacements for the CVP group. The predicted viability of a 2-cyanoindolizinyl alternative was confirmed experimentally and provided compounds with 0.4 nM activity against the wild-type virus. The compounds also performed well with EC₅₀ values of 10 nM against the challenging HIV-1 variant that contains the Lys103Asn/Tyr181Cys double mutation in the RT enzyme. Indolyl and benzofuranyl analogues were also investigated; the most potent compounds in these cases have EC₅₀ values toward wild-type HIV-1 near 10 nM and high-nanomolar activities toward the double-variant. The structural expectations from the modeling were much enhanced by obtaining an X-ray crystal structure at 2.88 Å resolution for the complex of the parent 2-cyanoindolizine <b>10b</b> and HIV-1 RT. The aqueous solubilities of the most potent indolizine analogues were also measured to be ∼40 μg/mL, which is similar to that for the approved drug efavirenz and ∼1000-fold greater than for rilpivirine

Bifunctional Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Mechanism and Proof-of-Concept as a Novel Therapeutic Design Strategy

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a major target for currently approved anti-HIV drugs. These drugs are divided into two classes: nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). This study illustrates the synthesis and biochemical evaluation of a novel bifunctional RT inhibitor utilizing d4T (NRTI) and a TMC-derivative (a diarylpyrimidine NNRTI) linked via a poly­(ethylene glycol) (PEG) linker. HIV-1 RT successfully incorporates the triphosphate of d4T-4PEG-TMC bifunctional inhibitor in a base-specific manner. Moreover, this inhibitor demonstrates low nanomolar potency that has 4.3-fold and 4300-fold enhancement of polymerization inhibition in vitro relative to the parent TMC-derivative and d4T, respectively. This study serves as a proof-of-concept for the development and optimization of bifunctional RT inhibitors as potent inhibitors of HIV-1 viral replication

Insights into DNA substrate selection by APOBEC3G from structural, biochemical, and functional studies

<div><p>Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis—for example, A3G deaminates the third dC in the CC<u><b>C</b></u>A sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1 nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, our biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.</p></div

P210 is important for selection of the -2 and +1 nucleotides at the A3G deamination sites.

<p>A) Relative single-cycle infectivity of VSV-G-psuedotyped HIV-1Δ<i>vif</i> viruses produced in the presence or absence of A3G-WT or A3G-P210R. Mean of two independent experiments done in triplicates are shown relative to the “no A3G” control (set to 100%); error bars, standard deviation. B and C) Effect of A3G-P210R substitution on the relative mutation frequencies at the +1 position nucleotide (b) and the -2 position nucleotide (c) with the preferred C at the -1 position (5’C<u><b>C</b></u>) or the non-preferred T at the -1 (5’T<u><b>C</b></u>) position. B) In the + 1 position nucleotide, mutation frequencies for the preferred sites with a C at -1 position (5’C<u><b>C</b></u>), A3G-WT prefers C<u><b>C</b></u>A or C<u><b>C</b></u>G over C<u><b>C</b></u>T and C<u><b>C</b></u>C; A3G-P210R prefers C<u><b>C</b></u>C over C<u><b>C</b></u>A, C<u><b>C</b></u>G, and C<u><b>C</b></u>T, but has no significant difference in preference between C<u><b>C</b></u>A and C<u><b>C</b></u>T. For the non-preferred sites with a T at -1 position (5’T<u><b>C</b></u>), A3G-WT and A3G-P210R both prefer T<u><b>C</b></u>A over T<u><b>C</b></u>T, T<u><b>C</b></u>G, or T<u><b>C</b></u>C. C) In the -2 position nucleotide, mutation frequencies for the preferred sites with a C at -1 position (5’C<u><b>C</b></u>), both A3G-WT and A3G-P210R prefer CC<u><b>C</b></u> over TC<u><b>C</b></u>, AC<u><b>C</b></u>, or GC<u><b>C</b></u>. For the non-preferred sites with a T at the -1 position (5’T<u><b>C</b></u>), A3G-WT prefers CT<u><b>C</b></u> over TT<u><b>C</b></u>, AT<u><b>C</b></u>, or GT<u><b>C</b></u>, whereas A3G-P210R prefers TT<u><b>C</b></u> or GT<u><b>C</b></u> over AT<u><b>C</b></u> and CT<u><b>C</b></u>. The significantly preferred nucleotide in the -2 or +1 positions are indicated (*<i>P <</i> 0.001). The number of sites, number of mutations, and relative mutation frequencies are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195048#pone.0195048.s002" target="_blank">S1 Table</a>.</p

Structure of Pot1A3G_CTD with ssDNA.

<p>A) Schematic of the Pot1A3G_CTD fusion protein design. Pot1 (pink) is fused directly to the N-terminus of A3G_CTD (blue). The ssDNA contains both Pot1 and A3G binding sites: the Pot1 site in dark gray and the A3G hotspot in light gray with the linker sequence in smaller font. The resolved adenine in the -1 pocket is colored orange and the expected deaminated cytidine is blue. B) Size exclusion binding test shows that Pot1A3G_CTD binds to the ssDNA substrate. Pot1A3G_CTD alone is in black, the ssDNA is in gray, and the mixture of the two is in red. C) Deamination activity using a UDG-dependent cleavage assay. The Pot1A3G_CTD fusion protein has the same deamination activity as that of A3G_CTD. D) Schematic and structure of the Pot1A3G_CTD in complex with DNA as observed in the crystal. The dA nucleotide bound to the -1 pocket is shown in orange. Two copies of the complex observed in the asymmetric unit are shown in blue (A3G), pink (Pot1), and grey/orange (DNA). The red star (schematic) and red sphere (structure) represent the zinc ion found in the catalytic site. The inset shows the 2Fo-Fc density (1σ contour level) observed for the adenine in the -1 nucleotide-binding pocket.</p